Neutrophil extracellular traps and it's possible role in cardiac pathology. The experience of cocultivation of neutrophils and cardiomyocytes. The role of proteasomal proteolysis in NETs formation.

1. Neutrophil extracellular traps in
pathology:
focus on heart diseases
Bogomoletz Institute of Physiology,
Kiev, Ukraine
Department of General and
Mollecular Pathophysiology
Vasyl Nagibin,
MD,PhD

2. Neutrophils
Young polymorph
nuclear (PMN)
Microphagocytosis
Enzyme exocytosis
Necrosis
Apoptosis
Inflammation in tissues
Normaly

3. • Neutrophil extracellular traps are
formed as the result of specific type of
programmed cell death called NETosis.
• The term “ETosis” is more wide and
used to determine this type of cell
death in other granulocytes
(eosinophils etc.)

4. The study of neutrophils: sensation after
more the 100 years of investigation.
Ilya Ilyich Mechnikov (1845-1916),
Nobel prize in 1908.
Arturo Zychlinsky
Discovered NETs in 2004,

5. Components and triggers of NETs
Extracellar trap components
DNA
Neutrophil Elastase
Histones
Myeloperoxidase
Lactoferrin
MMP9/Gelatinase B
Cathepsin G
Triggers of NETs formation
Et cetera
Рhorbol 12-myristate 13-acetate (PMA)
Activator of PKC, proinflammatory agent
LPS
IFNg
IL-8

6. NET in pathology
- antibacterial defense
- antiviral defense
- anticancer defense
- autoimmune diseases (systemic lupus erythematosus,
rheumatism, psoriasis);
- metastasis
- secondary alteration at inflammation of different
genesis
myocardial infarction

7. Myocardial infarction and NET
“There is an
inflammation, lets
go there”
Proinflammatory
Normal factors
conditions
Myocardial
infarction
Situation after myocardial
infarction
The goal of the work:
Can NETs provoke secondary
alteration at MI?
How to regulate it?

8. Materials and Methods
Isolation of neutrophils from rat blood,using Percoll gradient
Cultivation of neutrophils in RPMI w/o Phenol red and serum for 3
hours with or without (control) PMA (5 nM)
Measurment of DNA released (PicoGreen, fluorescence measurment),
and other NETs components (myeloperoxidase, immunohistochemistry)
(A.Zychlinsky et al; JCB,Vol176,N2,2007)
AND
Isolation of rat neonatal cardiomyocytes using Collagenase II and
Tripsin
Cocultivation of rat neonatal cardiomyocytes with neutrophils
Use of specific proteasome inhibitor (clasto-Lactacystin β-lactone) in
dose of 5 nM

9. DNA concentration in media after cultivation of
200 000 neutrophils without (control) or with
PMA
35
30
25
20
15
10
5
0
control PMA
% of DNA released

10. Fluorescent microscopy of PMN using Hoechst dye
control PMA
PMA
PMA was used for activatio9n of
NETs formation in dose 5 nM for 3
hours
Hoechst was used for detection of
DNA at fluorescent microscopy
40x

11. Fluorescent microscopy of PMN using Hoechst dye
control PMA
cL
PMA+cL
and anti-Myeloperoxidase Ab

12. The percentage of neutrophils with NET
under different conditions
120
100
80
60
40
20
0
Control
PMA
cL
PMA+cL
percentage of cells, %
live
foul of NET
NET

13. Coculture of rat neonatal cardiomyocytes and PMN
Hoehst 3334, propidium iodide
40 x

14. The percentage of necrotic cardiomyocytes in coculture
with PMN at different conditions of NETs formation (%)
30
25
20
15
10
5
0
контроль PMA лактацистин PMA+лактацистин

15. The percentage of necrotic cardiomyocytes in coculture
with PMN at different conditions of NETs formation after
anoxia (%)
60
50
40
30
20
10
0
контроль PMA лактацистин PMA+лактацистин

16. Thanks for some of my colleagues,
participating in this project
Prof. Moibenko O.O.
Prof. Dosenko V.E.
MD. PhD. Pashevin D.O.
BD. Tumanovska L.V.